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Melatonin induces transcriptional regulation of Bim by FoxO3a in HepG2 cells
S Carbajo-Pescador1, C Steinmetz, A Kashyap
1Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) and Institute of Biomedicine, University of León, León, Spain.
Background:
Melatonin induces apoptosis in many different cancer cell lines, including hepatocellular carcinoma cells. However, the responsible pathways have not been clearly elucidated. A member of the forkhead transcription factors' family, FoxO3a, has been implicated in the expression of the proapoptotic protein Bim (a Bcl-2-interacting mediator of cell death). In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate whether melatonin treatment induces Bim through regulation by the transcription factor FoxO3a.
Methods:
Cytotoxicity of melatonin was compared in HepG2 hepatoblastoma cells and primary human hepatocytes. Proapoptotic Bim expression was analysed by reverse transcriptase-polymerase chain reaction and western blot. Reporter gene assays and chromatin immunoprecipitation assays were performed to analyse whether FoxO3a transactivates the Bim promoter. Small interfering RNA (siRNA) was used to study the role of FoxO3a in Bim expression. Immunofluorescence was performed to analyse FoxO3a localisation in HepG2 cells.
Results:
Melatonin treatment induces apoptosis in HepG2 cells, but not in primary human hepatocytes. The proapoptotic effect was mediated by increased expression of the BH3-only protein Bim. During melatonin treatment, we observed increased transcriptional activity of the forkhead-responsive element and could demonstrate that FoxO3a binds to a specific sequence within the Bim promoter. Furthermore, melatonin reduced phosphorylation of FoxO3a at Thr(32) and Ser(253), and induced its increased nuclear localisation. Moreover, silencing experiments with FoxO3a siRNA prevented Bim upregulation.
Conclusion:
This study shows that melatonin can induce apoptosis in HepG2 hepatocarcinoma cells through the upregulation of proapoptotic Bim mediated by nuclear translocation and activation of the transcription factor FoxO3a.
Insights
Melatonin induces apoptosis in liver cancer cells by increasing the proapoptotic protein Bim. This occurs through the activation and nuclear translocation of the transcription factor FoxO3a.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Melatonin is known to induce apoptosis in various cancer cell lines, including hepatocellular carcinoma.
- The precise molecular pathways underlying melatonin-induced apoptosis remain incompletely understood.
- The transcription factor FoxO3a is linked to the expression of Bim, a proapoptotic protein.
Purpose of the Study:
- To investigate if melatonin treatment induces Bim expression via FoxO3a regulation in human HepG2 liver cancer cells.
- To elucidate the role of FoxO3a in mediating melatonin's proapoptotic effects in hepatocellular carcinoma.
Main Methods:
- Comparative cytotoxicity analysis of melatonin in HepG2 cells and primary human hepatocytes.
- Analysis of Bim expression using RT-PCR and Western blot.
- Reporter gene assays, chromatin immunoprecipitation, and siRNA experiments to assess FoxO3a's role in Bim transactivation and expression.
- Immunofluorescence to track FoxO3a localization.
Main Results:
- Melatonin induced apoptosis in HepG2 cells but not in primary hepatocytes.
- Increased expression of the proapoptotic protein Bim mediated the effect.
- FoxO3a was shown to bind the Bim promoter, and its nuclear localization and transcriptional activity increased upon melatonin treatment.
- Silencing FoxO3a abolished Bim upregulation.
Conclusions:
- Melatonin induces apoptosis in HepG2 hepatocarcinoma cells.
- This effect is mediated by the upregulation of the proapoptotic protein Bim.
- Melatonin achieves this through the nuclear translocation and activation of the transcription factor FoxO3a.
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